Physicochemical modification and application of alginate gels for the controlled release of reagents in classical solution assays and microfluidic assays

ABSTRACT

A reagent assay having a tubular member having a cross-section. The assay further has a first porous alginate plug shaped having the cross-section within the tubular member and containing a first reagent and a second porous alginate plug shaped having the cross-section adjacent the first porous alginate plug within the tubular member and containing a second reagent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a divisional of U.S. patentapplication Ser. No. 14/710,567 entitled Physicochemical Modificationand Application of Alginate Gels for the Controlled Release of Reagentsin Classical Solution Assays and Microfluidic Assays and filed on May12, 2015, which claims priority to U.S. Provisional Patent ApplicationSer. No. 61/992,119 entitled Reagent Assay System filed on May 12, 2014and to U.S. Provisional Patent Application Ser. No. 62/022,519 entitledLiquid Gellation [LIQ-GEL] Method to Enable Collection, Storage andAssay of Reagents or Analytes, filed on Jul. 9, 2014, all of which areincorporated herein by reference.

BACKGROUND

Alginate is an anionic polysaccharide harvested globally from elevenbrown seaweeds. The North American variety Macrocystis pyrifera (giantkelp) and other varieties are found spanning the globe in both thenorthern and southern hemispheres, with the only cultivar, Saccharinajaponica being developed in China, Japan, Russia, France and Korea. Inindustry, alginate applications of economic importance are in the fieldsof: dentistry (dental impressions), in the paint industry(plasticizers), in the food industry (as thickeners) and in the field ofpharmacopeia (as compounding agents for tablets, capsules and as activeingredients in upset stomach preparations). It is also used extensivelyin burn trauma units as a protective dressing that requires no adhesiveand subsequently is less painful for the burn patient on removal. It hasalso most recently gained fame in the theatrical and television industrywhen used by prosthetic makeup artists in the creation and modificationof science fiction characters and masks.

Alginate hydrogel contains a co-polymer of β-D-mannuronate (M) and itsC-5 epimer α-L-guluronate (G) that form a mixture of homopolymerconsecutive blocks (M or G) or alternating blocks (M-G-M-G- . . . ) orunequal mixtures of these blocks (M-M-G-M-G-G-M . . . ) may occur and isbased upon the prevalence of each co-polymer within the seaweed itself.An example representation of the structure of co-polymer components ofalginate is shown with reference to FIG. 1

As an example, variation occurs in the M to G ratio (M:G) based on theturbulence of the waters whence the brown seaweed was harvested. Inturbulent waters greater flexibility and higher strength of the seaweed(higher M content) is needed to maintain anchorage by a strong holdfast(analogous to a root in plants) and flexible stipe (analogous to a stem)as compared to a tidal pool that is sheltered. In this circumstance, thestipe may require more structure (higher G content) to enable the blade(analogous to leaves in plants) to rise above the water surface enablingsuccessful competition against green algal mats that often cover thewater surface.

Other modifying factors are the temperature and the depth of the watersfrom where it was harvested, the age of the kelp and whether theyharvested the entire kelp holdfast, stipe or blade or only a portion(e.g.: the blade). In each case, the living kelp produces theappropriate M:G ratio to maintain its ecosystem. In some cases an algalkelp forest may include other kelp species that are harvestedunintentionally (some giant kelp forests include 20 other species thatare largely undisturbed when only the giant kelp blade is harvested).These factors promote variation from one alginate product lot to thenext and therefore a small and simple test is required to select theappropriate ionic strength concentration (salts of sodium, potassium andcalcium) for creating the desired porosity. From that point forward,constancy of formulation within the lot remains the same. This is acommon practice in most biological reagents as the “activity of enzymes”changes with each lot. Alginates are available and sold in United StatesPharmacopeia (USP) grade that requires sufficient purity to meet orexceed that used for food, drug or medicinal use.

SUMMARY

A reagent assay in accordance with the present disclosure has a porousalginate core containing a first reagent, a first polysaccharide layercoating the alginate core and having a second reagent, and at least onepolysaccharide core comprising a hydration seal and coating the firstpolysaccharide layer.

BRIEF DESCRIPTION OF THE DRAWINGS

The various embodiments of the present disclosure are best understood byreferring to FIGS. 1 through 6 of the drawings. The elements of thedrawings are not necessarily to scale, emphasis instead being placedupon clearly illustrating the principles of the disclosure. Throughoutthe drawings, like numerals are used for like and corresponding parts ofthe various drawings.

FIG. 1 is a representation of the molecular make-up of alginate.

FIG. 2 is a graph showing absorbance versus wavelength of various geland gum materials.

FIG. 3 depicts a prepared bead for reagent assay in accordance with anembodiment of the present disclosure.

FIG. 4 depicts enhanced porosity of alginate in alginate samples.

FIG. 5 depicts another reagent assay in accordance with anotherembodiment of the present disclosure.

FIG. 6 depicts an alginate lyase.

DETAILED DESCRIPTION

The present disclosure describes a composition of a porous alginate andmethod of chemically modifying the porosity and conductivity of alginatethat can in tandem with other degradable polysaccharides be used in thecontrolled release of reagents for chemical assays. By modification ofalginate with certain solubilized ionic salts, various degrees ofporosity can be generated in the hydrogel. The porous hydrogel may beused as a filtering device. By addition of solutions of electrolytes andoxides the conductivity of the alginate hydrogel can be increased ordecreased, respectively. The porous alginate can incorporate a reagentused for the determination of an analyte. The reagent releases onexposure to the reacting solution. The porous alginate can be used as ahydrogel core for a series of coatings of reagents mixed with variousmolecular weight polysaccharides. These are designed to release thereagents serially from the outer layer to subsequent interior andfinally inner hydrogel core layers, based on the decreasing solubilityof each polysaccharide. If the analyte is sensitive to dilution, theselected polysaccharide(s) can be solubilized with the specificaffiliated lyase. The catalytic mechanism, via the breaking ofglycosidic bonds in the polysaccharides increases the solubility withnegligible dilution of the analyte sample. Alginate hydrogel has twoaffiliated lyases. This enables implantation of toxic or catalyticmaterials in the hydrogel, minimizing exposure of technical personnelwhile permitting recycling and recovery of the catalytic material at anappropriate facility.

Alginate, once gelled does not solubilize on further dilution with thesame solvent (in contrast to the other mentioned high molecular weightpolysaccharides that dissolve in excess solvent). Alginate can have gelsetting times of 1-20 minutes and this is a function of the salt used,the dilution factor and set temperature. For the purpose of designingalginate of varying porosity a longer setting time of at least 10minutes is desirable. To achieve the desired setting time a dilutionfactor of 1:5 parts (dry weight of alginate plus added reagents tosolvent weight of distilled water). This enabled homogeneous mixing ofthe gel during the workable stage to a smoother consistency and time forpipetting of various volume droplets (in these experiments 50-300microliter beads of initial volume were produced) onto Teflon bakingsheets or wax paper prior to being fully set. This mixing and droplet(bead) forming process can be easily automated with existing industryequipment. Alternately larger gel samples were prepared by placing thesamples in 1 mL or 5 mL pipet tips. Once fully set alginate undergoessyneresis. This is the process that sparked interest in theinvestigation of alginate for reagent delivery and led to subsequentother discoveries and applications. Syneresis is the expulsion of liquidfrom the set gel as it undergoes contraction. The gelled droplets sweat.

The first experiment performed was the coating of prepared beads usingthe syneresized liquid from the bead to adhere a dry chemical reagent.It was found that further evaporative loss could be prevented by dustingthe reagent covered bead with confectioner sugar. Further investigationdemonstrated that other gum types were more suitable and stable as acoating seal material. These included, but are not limited to, gumarabic, locust bean gum, guar gum, carrageenan and gellan. UVSpectrophotometric analysis, a graph of which is shown in FIG. 2, ofthese polysaccharides showed that the absorbance of these species wasbelow 310 nanometers (nm) (in all samples except guar gum that continuedto show slight absorbance between 0.075 decreasing to 0.015 at 390 nm)and therefore insignificant and well below the range of observation forthe testing of drinking and waste water quality—the first targetapplications.

A second series of experiments were performed that demonstrated that asecond reagent could be added to the first reagent over the alginatecore, by mixing it in gellan and applying by a rolling process to theexisting bead. This could be done in a manufacturing process through aspray coating and drying process.

A third series of experiments demonstrated that by approaching the limitof gelation of alginate (higher ionic strength addition), in addition toprotracting the setup time, the alginate had greater porosity. By mixingthe added salts (source of cations) with high molecular weightdyes—specifically Allure (a red dye) and Blue Dextran at these higherionic strength solutions, it was found that the alginate matrix at theselected ionic strength did not retain these dyes. However in samplesmixed with lower concentration of salt, these dyes were entrapped withinthe resulting alginate hydrogel. The ionic strength desired to obtainhigh pore content were able to be visually determined by the includedindicator dye. When alginate at the specific ionic strength was mixedwith blue or red dye to produce a pale colored hydrogel, a highly porousgel matrix was indicated when a concentrated dye exudate appeared duringsyneresis. If the ionic strength was lower, the gels retained the palecoloration as the dye was entrapped within the gel. Thus, to producehigher porosity of the alginate was experimentally determined to be atthe limit that maintained larger gel pores capable of exuding the bulkydye molecules. Upon soaking in solvent the entrapped dye in the lowerionic strength gel mixtures eventually diffused from the colloidindicating that the retention time of the dye was very protracted due tosmaller pore size.

Disclosed is a method of chemically modifying the porosity as depictedin FIG. 4 and that can in tandem with other degradable polysaccharidesbe used in the controlled release of reagents for chemical assays asshown in FIG. 3. By modification of alginate with certain solubilizedionic salts, various degrees of porosity can be generated in thehydrogel. The hydrogel formulation contained 92% to 98% weight to totalweight (% w/w) of dry sodium alginate with up to 8% added salt. Notethat the percentages of substances is for exemplary purposes and otherpercentages of substances may be used in other embodiments. This rangereflects the use of two product sources of sodium alginate. One sourceis a prosthetic grade sodium alginate, a product from harvested brownseaweed (kelp), and the other source was Reagent Grade Alginic AcidSodium Salt by ACROS (CAS 9005-38-3—Lot A0339825). A matrix screen wasconducted with various additions of sodium, calcium, or potassium saltsat ambient temperature (68° F./20° C.) or slightly below ambienttemperature. The result of the matrix screen indicated that thepotassium cation from potassium chloride (KCl from Fisher Scientific525484A Lot 4AH1307-3125A) and potassium iodide (KI from ACROS CAS7681-11-0 Lot B0137921) produced large pores that retained uniformity.The amount of the potassium iodide required for the pore formation was4% w/w when using the prosthetic grade alginate as depicted in FIGS. 4.400 and 401 (repeat sampling) that shows uniformity in the matrixscreen. The ACROS product also produced uniform pore formation ofcomparable size at 7.5% w/w. Greater addition of the selected salts wereneeded when using the ACROS product or Fisher product, presumably as aresult of product higher purity. The alginate once gelled remainedstable and pliable for several months when stored in a container ofminimal air volume to maintain humidity level. This enables storage ofprepared beads.

FIG. 3 depicts a prepared bead 300 in accordance with an embodiment ofthe present disclosure. The prepared bead 300 comprises an inner porousalginate core 301 created by modification of alginate hydrogel, asdescribed further herein, and containing a first reagent A. Note thatthe identification of reagent A in the alginate core 301 is forexemplary purposes only, and greater or fewer occurrences of reagent Aare possible and probable. The prepared bead 300 shows the porous coreon which additional polysaccharide plus reagent layers may be addedcomprising outwardly extending layers as shown in FIG. 3. The outerlayer 304 is solely a polysaccharide used as a seal.

As noted hereinabove, by modification of alginate hydrogel via inclusionof various percentages by weight (hereinafter “% w/w”) of sodium,calcium and potassium salts that introduce porosity within the hydrogel,an analyte assay reagent may be incorporated within these pores andsubsequent polysaccharide coating layers and be released on exposure toaqueous solution or solvent. Note that the hydrogel formulation contains92% to 98% weight to total weight (% w/w) of dry prosthetic grade sodiumalginate. The prepared salts of alginic acid (sodium alginate, potassiumalginate or ammonium alginate) form an irreversible hydrocolloid gelmatrix in comparison to the non-water soluble alginic acid. Thereforethe prepared salts are the base formulation used in these compositionsprior to addition of further salts used to generate the pore formation.Addition of the analytical reagents may be mixed into wet pre-setalginate, adhered to the surface of set alginate, incorporated intodehydrated dried porous alginate in reagent solution under vacuum ortumbled as a dry reagent into dry porous alginate beads.

In accordance with an embodiment of the present disclosure, a serializedtimed-release of reagents, e.g., reagents A, B, and C depicted in FIG.3, can be achieved by encapsulating reagents within and in surroundinglayers of a porous hydrogel core, as described hereinabove. As shown,reagents may be layered on the alginate core 301 separated by a seriesof polysaccharides layers 302 and 303 of successively lower solubilityas the layers 302 and 303 move outward from the core. Note that eachreagent A, B, and C would be mixed with a gum agent that includes but isnot limited to locust bean gum, guar gum, gum arabic, tara gum, xanthangum, pullulan gum and gellan gum. The molecular weight composition andaffiliated solubility of these gums in aqueous solution vary. Reagentsare solubilized by exposure to aqueous solution until all of thereagents have reacted in the analyte assay sequence, minimizing thereagent quantities needed, maximizing reaction efficiency andeliminating competing reactions that occur in alternate bulk solutionprocesses. As shown in FIG. 3, the porous core 301 contains the lastreagent for release in the analyte process and below the outerpolysaccharide seal is the first reagent C to be released. Each of thereagents C, B and A would be released by dissolution of the previouslayer.

In one embodiment, use of lyase 600 as shown in FIG. 6 in the assayreduces the amount of solvent (usually water) to dissolve thepolysaccharide-reagent containing layers 302-303 shown in FIG. 3. Ifdilution by aqueous medium of the analyte is problematic, each of thelong chain polymer polysaccharide gums selected can be enzymaticallycleaved into smaller segments by lyase 600. Note that lyases arespecific for cleavage of a particular polysaccharide (e.g: a lyase forgellan would be different than one for alginate). The mechanism ofaction is usually through cleavage of the glycosidic bond through a(3-elimination process. By breaking these long chain polymers into smallchains, solubility is enhanced. As a catalytic material, lyase addition(usually by droplet) is a small volume addition. As an enzymatic proteincatalyst a spectrophotometric peak will be evident at 235 nm. This UVregion is usually below the analyte test region for conventional wateror wastewater analysis—however it may interfere with certain biologicaltesting assays centering on 235 nm.

Note that in one embodiment, the porous hydrogel without the inclusionof reagents may be used as a filtering device or size exclusion devicein columns, funnels, pipets and microfluidic channels.

FIG. 5 depicts another exemplary reagent assay 500 in accordance with anembodiment of the present disclosure. The reagent assay 500 comprises acontainer 505, which may be for example an open-ended pipet. Inaddition, the reagent assay 500 comprises one or more porous alginateplugs. In this regard, the reagent assay 500 comprises porous alginateplugs 502-504.

As noted hereinabove, each porous alginate plug 502-504 is made up of aporous alginate substance. Further, within the porous alginatesubstances are respective reagents A, B, and C. Note that only a singleletter, including A, B, and C, are shown for exemplary purposesdepicting the reagent. However, each porous alginate substance includesa myriad of instances of the reagent contained therein.

In use, an analyte solution, depicted by reference arrow 506, is pouredinto the container 505 in the fluid reservoir 501. From there, theanalyte solution 506 travels through the first porous alginate plug 502reacting with reagent A. Thereafter, the analyte solution 506 travelsthrough porous alginate plug 503 reacting with reagent B, then throughporous alginate plug 504 reacting with reagent C. Once the analytesolution 506 has traveled through each porous alginate plug, the reactedanalyte solution, depicted by reference arrow 507, may exit the tube forfurther analysis.

In such an embodiment, a series of tubular slugs of alginate containingreagents can be aligned and placed in tandem within a cylindrical tube.The analyte can be placed at the opening of the tube and permitted topercolate through each successive slug. The solution exiting the tube(outflow) would then be ready for the detection method for that analyte.This could also be readily adaptable to microfluidic methods where thegelled reagents are placed inline in the microfluidic channels. Thisenables the sealing of a microfluidic chip with all reagents constrainedwithin an insoluble and immobilized gel. This type of microfluidic chipcould function in gravity driven, applied pressure or in sipper mode(pulling of the solution by gentle suction on the outflow port). Forutilization of this method in the field, a tube (straw-like) couldinclude the gel slugs with reagents, the analyte, for example a watersample, could be guided gently through the tube (using a syringe bulb)and the sample collected for detection. If the method is colorimetricthis could be compared to a color chart.

Note that in one embodiment the porous alginate core 301 (FIG. 3) may beused as a non-dissolving substrate to contain catalytic materials thatmay be reused or recovered for recycling. This sustainable method alsopromotes safety in handling. The alginate core is an irreversiblehydrocolloid gel. In processes requiring rare or toxic catalystinclusion, the ability to recover these materials is important. As anexample, cadmium beads within a glass column are often used in reductionassays. Cadmium beads could be mixed with porous alginate, confining itfor safety within the gel (breakage of a glass column would not causecadmium spillage as it would remain intact in the hydrogel). Inmicrofluidic applications where reagents are stored on a chip thecatalytic material could be extracted via a punch mechanism andrecycled. Alginate also has two specific lyases. One breaks theglycosidic bond of an M-block and the other breaks the glycosidic bondof a G-block. Therefore addition of these lyases to alginate, andsubsequent disruption of those bonds enables extraction of the cadmiumbeads on sedimentation or sedimentation-assisted centrifugation.

What is claimed is:
 1. A reagent assay, comprising: a tubular memberhaving a cross-section; a first porous alginate plug shaped having thecross-section within the tubular member and containing a first reagent;and a second porous alginate plug shaped having the cross-sectionadjacent the first porous alginate plug within the tubular member andcontaining a second reagent.
 2. A method of modifying alginate,comprising: providing a sample of porous alginate; and mixing in thealginate a reagent that is encapsulated by one or more pores in thealginate.